Messenger RNA untranslated regions (UTRs) play major roles in regulating gene expression. Many 3' UTR cis-regulatory elements, including microRNA targets and AU-rich elements, have been identified using chemical cross-linking methods and computational predictions. Most of the known elements have been discovered from low throughput experiments, which are slow, expensive, and heavily biased. Further work is necessary to identify novel cis-regulatory elements, test the activity of computationally predicted elements, and discover trans-regulatory factors required for 3' UTR activity in cells. The Erle and McManus laboratories have been working together to generate new tools for systematic functional analysis of 3' UTR sequences and for genome-wide shRNA screens in human cells. We developed a high-throughput assay for efficient Massively Parallel Identification of Regulatory Elements in UTRs (eMPIRE). The eMPIRE assay utilizes a novel tetracycline-regulated lentiviral reporter construct, massively parallel array-based oligonucleotide synthesis, flow cytometric sorting, and high- throughput sequencing. In addition, we developed an EXPANDed genome-wide lentiviral shRNA library that will allow us to rapidly identify cellular factors that are required for UTR regulatory effects. We present preliminary data establishing that these novel methods are powerful tools for identifying cis- regulatory 3' UTR sequences and trans-regulatory factors in human cells. No other technology offers the ability to perform this kind of quantitative discovery for functional genetic in mammalian systems. This project will generate comprehensive data about the functional activity of sequences of all human 3' UTRs, refine computational methods for predicting the functional activity of 3' UTR sequences, identify novel 3' UTR cis-regulatory elements and motifs, and reveal many genes and pathways that mediate 3' UTR functions in human cells. This is made possible via collaboration between two collaborating labs that have developed the appropriate high-throughput tools to successfully accomplish the proposed plan. Information from this project will be used to annotate genomic databases, providing mechanistic insight into potential human polymorphisms. Likewise, it will lay an incredible foundation for further understanding post-transcriptional gene regulation governed by sequence elements.